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DNA Normalization

A crucial laboratory protocol in molecular biology and proteomics, used to ensure consistent concentrations of components (like DNA, RNA, or protein) across samples. It enhances the accuracy and reliability of downstream analyses, such as sequencing, PCR, or protein assays.

Concentration Normalization 1

DNA normalization (concentration normalization) is typically used in workflows like Next-Generation Sequencing (NGS), Polymerase Chain Reaction (PCR), and proteomics. Despite its importance, DNA normalization can be difficult and prone to errors due to the need for precise measurements and calculations, especially when using manual pipetting.

Manual normalization, normalization using a liquid handling system, and normalization using a microplate reader are some methods employed for this process. However, automated systems are increasingly preferred due to their higher accuracy, reproducibility, and efficiency, as well as their reduced risk of contamination.

Workflows Needing DNA Normalization

The technique is commonly used in a variety of molecular biology workflows, including:

  1. Next-Generation Sequencing (NGS): In NGS, concentration normalization is essential to ensure that each sample contributes equally to the sequencing run.
  2. Polymerase Chain Reaction (PCR): Before performing PCR, DNA samples are often normalized to ensure equal amplification across all samples.
  3. Proteomics: For protein assays, it’s important to have equal concentrations of protein across all samples to ensure accurate comparison and analysis.

DNA normalization has never been easier

The OT-2 is a bench-top liquid handler designed to be accessible and flexible enough to automate many common applications.

Top Methods of Performing Concentration Normalization

  1. Manual Normalization: This involves using a spectrophotometer or a fluorometer to measure the concentration of DNA, RNA, or protein in each sample, and then manually diluting or concentrating each sample to reach the desired concentration.
  2. Normalization Using a Liquid Handling System: This automated method uses a liquid handling system to measure and adjust the concentration of each sample. This is usually more precise and less prone to error than manual normalization.
  3. Normalization Using a Microplate Reader: This method involves using a microplate reader to measure the concentration of each sample in a multi-well plate, and then a liquid handling system to adjust the concentrations.

Protocol Spotlights

Opentrons helps you automate DNA normalization with open-source protocols for the OT-2 and Opentrons Flex

Why is DNA Normalization So Difficult?

DNA normalization is difficult because it requires precise measurements and calculations. Even a small error can significantly affect the final results. It is also time-consuming and laborious, especially when dealing with large numbers of samples.

Key Challenges of Concentration Normalization with Manual Pipetting

Manual pipetting poses several challenges, including:

  1. Human Error: Manual pipetting is prone to human error, which can lead to inaccuracies in the final normalized concentrations.
  2. Reproducibility: Due to slight variations in technique among different lab personnel, it can be difficult to achieve consistent results with manual pipetting.
  3. Efficiency: Manual pipetting is time-consuming, especially when dealing with large 

numbers of samples.

How to Automate the DNA Normalization Process

The normalization process can be automated using liquid handling systems. These systems can measure the concentration of each sample using spectrophotometry or fluorometry, calculate the required adjustment, and then accurately dispense the appropriate amount of diluent or concentrated sample to achieve the desired concentration.

Benefits of Automation over Manual Pipetting for DNA Normalization

Automation of the normalization process offers several advantages over manual pipetting, including:

  1. Accuracy: Automated systems are generally more precise and less prone to error than manual pipetting.
  2. Reproducibility: Because the process is automated, it can be reproduced exactly the same way each time, leading to more consistent results.
  3. Efficiency: Automated systems can process many samples simultaneously, significantly reducing the time required for DNA normalization.
  4. Reduced Risk of Contamination: Automated systems minimize the need for human interaction, thus reducing the risk of sample contamination.

Want to know more?

Our team of experts can help figure out if automation is right for you. Book a virtual demo to discuss your workflow needs with an expert.